Method of producing fusion proteins

ABSTRACT

Disclosed are methods of producing fusion proteins including those with dual biological activities. These methods include the provision of a first and second DNA sequence encoding a first and second polypeptide, repectively, the digestion of the first DNA sequence at a restriction site adjacent its 3&#39; or 5&#39; terminus, and the ligation of a linker/adapter sequence (l/a) to the restricted end of the first DNA sequence, thereby forming a cassette. The l/a includes, at one end, that portion of the first DNA sequence extending from its terminus nearest the restriction site to the restriction site, and at the other end, one side of a splice site. A eucaryotic host cell is transfected with the cassette and the second DNA sequence having, at one end, one side of a splice site compatible with the side of the splice site on the l/a. The transfected host cell is cultured to express the transfected DNA as a single chain fusion protein.

This is a continuation of copending application Ser. No. 07/810,522filed on Dec. 17, 1991 now U.S. Pat. No. 5,196,320 which was acontinuation of application Ser. No. 07/409,889 filed on Sep. 20, 1989and which is now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to the production of recombinant polypeptides,and in particular, to the production of synthetic proteins having pluralfused domains. More specifically, this invention relates to methods ofproducing recombinant fusion proteins having dual biological activitiessuch as binding molecules and receptor proteins with preselectedspecificity and activity. Such fusion proteins are useful, for example,in imaging procedures, in the diagnosis and treatment of various humancancers, infectious diseases, and dysfunctions, and in the developmentof vaccines.

Fusion proteins having dual activities and/or functions are known andinclude combinations of peptide hormones, enzymes, transport andreceptor proteins, viral coat proteins, interleukins, lymphokines,immunoglobulins (Igs), and fragments thereof. Some of these fusionproteins are known to provide enhanced antigenicity, and therefore, areuseful in the production of vaccines. Others have high biologicalactivity, and hence, are useful in treating various infectious anddeficiency diseases and disorders. Still other fusion proteins areuseful as diagnostic agents because of their enhanced targetingabilities. (See, e.g., U.S. Pat. Nos. 4,223,270, 4,801,536, CA 1217156A,WO 8806630A, and JP 63267278A for examples of such fusion proteins.)

Immunoglobulin molecules have been produced as fusion proteins (e.g.,chimeric antibodies). Their structure is particularly conducive to theformation of fused polypeptides having a first protein domain (e.g., avariable region) from a first Ig molecule from one species having aparticular specificity, and a second domain (e.g., a constant region)from a second Ig of a different species (and perhaps specificity). Theywere developed as an alternative to non-chimeric monoclonal antibodies,many of which are of non-human (e.g., murine) origin, and hence may beantigenic to humans. Human monoclonal antibodies are the most desirabletherapeutic agents, as their use should provoke a greatly reduced immuneresponse in humans. However, the production of human monoclonalantibodies by cell fusion methodologies is difficult, as immunized humanspleen cells are not readily available, and as human hybridomas arenotably unstable.

Chimeric antibodies composed of both human and non-human amino acidsequences should elicit less of an immune response in humans, andtherefore should have improved therapeutic value. Accordingly, hybridantibody molecules have been proposed which consist of Ig light (L) andheavy (H) chain amino acid sequences from different mammalian sources.The chimeric antibodies designed thus far comprise variable (V) regionsfrom one mammalian source (usually murine), and constant (C) regionsfrom human or another mammalian source (see, for example, EPOapplication nos. 84302368.0 (Genentech), 85102665.8 (ResearchDevelopment Corporation of Japan), and 85305604.2 (Stanford University);P.C.T. application no. PCT/GB85/00392 (Celltech Limited); Morrison etal. (1984) Proc. Natl. Acad. Sci. U.S.A. 81:5851-6855; Boulianne et al.(1984) Nature 312:643-646; and Sahagan et al. (1986) J. Immunol.137:1066-1074).

The production of recombinant chimeric antibodies with predeterminedspecificity has typically involved the use of cloned genomic DNAfragments. For example, the genomic DNA sequences encoding H and Lchains can be cloned in their rearranged forms (i.e., in the DNAsequence that results from recombination events during B Cellmaturation). As such, these genomic sequences contain the informationnecessary for their expression, (i.e. the 5' untranslated sequences,promoter, enhancer, protein coding region, and donor splice site). Thedonor splice signals at the 3' end of the V gene segments are compatiblewith the splice acceptor signals at the 5' end of the Ig regions ofother species. That is, the splice product between the two maintains thecorrect reading frame. For example, when a murine V and a human C_(k)segment are joined and transfected into the appropriate host cell type,the primary transcript is correctly spliced and results in a maturemessenger RNA (mRNA) molecule with an open reading frame through boththe V and C regions.

There are disadvantages to the use of genomic V region fragments for theexpression of recombinant chimeric antibodies. The first involves thecloning process itself which can be quite laborious for single-copygenes, requiring the screening of many independent clones of a phagelibrary. Furthermore, many hybridomas contain multiple rearranged Vgenes which represent non-productive recombinational events. Theidentification of the expressed V_(L) or V_(H) segment can often requireextensive DNA sequence analysis as well as confirmation by cloning andsequencing the DNA copy of the expressed mRNA (complementary or cDNA).

A more direct approach is to clone the cDNAs for both the L and Hchains, and to use cDNA expression vectors for their expression. In thiscase, cloning is simple and rapid, since Ig mRNA is very abundant inhybridoma cells, and highly efficient methods for cDNA cloning areavailable. However, when one uses this approach, a problem arises whenthe separate expression of different V and C regions from different IgcDNAs is desired, as in the case of chimeric antibodies having, forexample, murine V regions and human C regions. The Ig cDNA represents adirect copy of the mRNA which, in turn, is a fusion of V and C exonsthrough normal, in vivo RNA splicing into a continuous polynucleotidesequence. Precise excision and recombination of a murine V_(H) with ahuman Cγ₁, for example, is not possible because appropriate restrictionsites are not present at the VC junction of both sequences.

The expression of chimeric antibodies has been accomplished through theuse of cloned cDNAs. This procedure may involve the mutagenesis ofsequences in both the murine V region and human C region, near the VCjunction, such that a common restriction site is created for directlyjoining the eDNA segments (Liu et al. (1987) Gene 54:33-40).

It is an object of this invention to provide a method of producing fusedpolypeptides. Another object is to provide methods for producing fusionproteins having dual activities and/or functions such as, for example,chimeric immunoglobulin molecules having a predetermined antigenspecificity. Another object is to provide a relatively simple and rapidprocedure for providing human/non-human mammalian chimetic antibodiesand truncated versions thereof having reduced antigenicity in the humanbody. Yet another object of the invention is to provide a rapid methodof producing a made-to-order chimetic immunoglobulin molecules utilizinga specifically engineered V region gene that can be attached easily toanother gene, and transfected and expressed in a host cell.

SUMMARY OF THE INVENTION

The present invention provides methods of general applicability forproduction of fusion proteins including at least a portion of an enzyme,peptide toxin, peptide hormone, lymphokine, hormone, interleukin, and/orimmunoglobulin. The invention further provides for the modification ofcloned cDNAs such as Ig cDNAs so that a polypeptide such as a V regionwith predetermined selectivity can be expressed as an independent unitor cassette fused with any one of a variety of polypeptides such as anIg C region from the same or a different species.

One new method involves the construction of a linker/adapter (l/a)sequence which enables the fusion of a DNA sequence encoding a firstpolypeptide having a naturally occurring restriction site at or near its3' terminus, to a DNA sequence encoding a second polypeptide. The l/asequence includes, in sequence from its 5' end, a sequence of DNAencoding the first polypeptide from a (near-)3' terminal restrictionsite to its 3' terminus, and a splice donor site compatible with asplice acceptor site on the 5' terminus of DNA encoding a secondpolypeptide. In a preferred embodiment, the l/a includes a universalintron having greater than 80 nucleotide bases in length.

To produce the fusion protein, the DNA sequence encoding the firstpolypeptide is digested at a suitable restriction site near the 3' endof the gene sequence. The 5' end of the l/a is then ligated to thatsite, thereby producing a cassette with a splice donor site at its 5'end. A eucaryotic host cell is transfected with the cassette as well aswith a second DNA sequence encoding a second polypeptide and having thecorresponding splice partner site (splice acceptor) at its 5' terminus.The transfected cell is then cultured to co-express the cassette and thesecond DNA sequence as a single chain fusion protein.

Alternatively, a first DNA sequence encoding a first polypeptide may bedigested at a restriction site located 3' to and adjacent its 5'terminus, thereby producing a new 5' end of that first DNA sequence. Al/a is then ligated to the new 5' end of the first DNA sequence. Thisl/a includes, in sequence from its 3' end, DNA encoding that portion ofthe first polypeptide extending from a (near-)5' terminal restrictionsite to its 5' terminus, and a splice acceptor site compatible with asplice donor site on the 3' terminus of the DNA encoding a secondpolypeptide. In a preferred embodiment of the invention, the l/aincludes greater than about 80 nucleotide bases in length.

To produce the fusion protein, the DNA encoding the first polypeptide isdigested at an appropriate restriction site near the 5' end of the gene.The 3' end of the l/a is then ligated to that restricted site, therebyproducing a cassette with a splice acceptor site at its 5' end. Aeucaryotic host cell is transfected with the cassette as well as with asecond DNA sequence encoding the second polypeptide and having thecorresponding splice partner site (splice donor) at its 3' terminus. Thetransfected cell is then cultured to co-express the cassette and thesecond DNA sequence as a single chain fusion protein.

In the case of the production of chimeric antibodies, the methodincludes the reconstruction of a cDNA, restricted at a naturallyoccurring restriction site unique to the V region-encoding portion ofthe cDNA, by ligation of a synthetically produced DNA sequence whichreplaces the removed 3' terminus of the V region and adds a 3' donorsplice site that mimics the donor splice site normally found on thegenomic V region-encoding DNA. This V region "cassette" can be used inconjunction with any C region-encoding DNA sequence, or otherprotein-encoding exon, having a complementary 5' splice acceptor site. AcDNA is synthesized using reverse transcriptase and a mRNA sequence asthe template which contains at least the mRNA sequence encoding the Ig Vregion. This V region-encoding DNA sequence includes a restriction sitelocated on the 5' side of, and adjacent, the junction (VC) of the Vregion sequence and the flanking sequence encoding a C region. Theunique restriction site is chosen to be a specific DNA sequence that isfound rarely or only once in the V region DNA, and which is recognizedby a particular restriction endonuclease capable of cleaving the DNA atthat sequence.

Digestion at the unique restriction site leaves a fragment which encodesat least most of the V region, and preferably defines the 3' end. To the3' end is ligated an oligodeoxynucleotide referred to herein as a l/asequence. The l/a sequence includes a DNA portion extending from the VCjunction 5'-ward to the restriction site. In addition, the l/a sequenceincludes at its 3' end a donor splice site which mimics the function ofthe splice site present in genomic V region-encoding DNA prior to thesplicing and rearranging events which occur in the nucleus during mRNAassembly. When ligated together, the restricted V region-encoding cDNAand the l/a sequence form what is referred to herein as a V regioncassette.

An appropriate host cell, such as a hybridoma or myeloma, is thentransfected with a vector containing the V region cassette and with DNAencoding at least a portion of a polypeptide, such as a C regionsequence, which includes a splice acceptor site at its 5' end and a stopsignal. The splice acceptor site is compatible with the splice donorsite on the 3' end of the V region cassette. The DNA sequence encodingthe second polypeptide may be readily retrieved from human genomiclibraries. Once isolated, it may be used repeatedly to manufacture Ighaving human C regions.

The transfected cell is then cultured to co-express the V regioncassette and the C region DNA. A clone which integrates the V regioncassette and the C region DNAs splices the separate V and C region DNAsto produce a mRNA encoding a synthetic fused protein comprising the fulllength V region and a C region or portions thereof. Cotransfection withconstructs encoding V_(H) and V_(L), suitably introduced in tandem withC_(H) and C_(L) sequences, express binding proteins having an authenticFv domain with binding properties similar to those of the original Igand C regions from a desired species.

In some embodiments, the resulting polypeptide includes an Ig V regionof murine origin and at least a portion of a human C region, therebyforming a chimeric Ig or fragment thereof. The term "chimeric Ig" isused herein to describe an Ig having amino acid sequences derived fromIgs of differing specificities and/or different species.

However, it should be appreciated that a given V region or portionthereof may be ligated in this fashion to polypeptides other than Ig Cregions to produce chimetic binding molecules other than Ig's.Furthermore, a V region need not be part of the resulting fusion proteinat all.

BRIEF DESCRIPTION OF THE DRAWING

The foregoing and other objects and features of the invention, as wellas the invention, itself, may be more fully understood from thefollowing description when read together with the accompanying drawingsin which:

FIGS. 1A and 1B are schematic representations of the methods of theinvention;

FIG. 2 is the nucleic acid sequence and deduced corresponding amino acidsequence of the murine monoclonal 14.18 H chain V region, including aleader sequence, V region, VC junction, and part of a C region;

FIG. 3 is the nucleic acid sequence and deduced corresponding amino acidsequence of the murine monoclonal 14.18 L chain V region, including aleader sequence, V region, VC junction, and part of a C region;

FIGS. 4 panels A-E schematically depict the construction of arepresentative H chain V region cassette: FIG. 4A is a schematicrepresentation of the murine H chain cDNA including a V and a C region;FIG. 4B is a schematic representation of a V region cassette includingthe V region cDNA sequence linked at the ScaI site to an l/a DNAsequence comprising a copy of the 3' end of the V region and a splicedoner site (ScaI-Hind III); FIG. 4C is the amino acid sequence andcorresponding cDNA sequence of a portion of the murine H chain genomicDNA extending from the unique restriction site (here Sca I) in the Vregion through the VC junction (indicated by a vertical line); FIG. 4Dis the nucleic acid sequence of the l/a comprising the portion of the Vregion sequence extending from the unique restriction site to the VCjunction, and including a "universal intron" having a splice donor siteadjacent the V region terminus; and FIG. 4E is a schematicrepresentation of the l/a;

FIGS. 5 panels A-E schematically depict the construction of arepresentative L chain V region cassette: FIG. 5A is a schematicrepresentation of the murine L chain cDNA including a V and C region;FIG. 5B is a schematic representation of a V region cassette includingthe V region cDNA sequence linked at the Afl III site to an l/a DNAsequence; FIG. 5C is the amino acid sequence and corresponding cDNAsequence of a portion of the murine L chain extending from the uniquerestriction site (here Afl III) in the V region through the VC junction(indicated by a vertical line); FIG. 5D is the nucleic acid sequence ofthe l/a comprising the portion of the V region DNA sequence extendingfrom the unique restriction site to the VC junction, and including a"universal intron" having a splice doner site adjacent the V regionterminus; and FIG. 5E is a schematic representation of the l/a;

FIGS. 6 panels A-C describe an exemplary cloning strategy for theconstruction of a chimeric Ig expression vector: FIG. 6A is a schematicrepresentation of the integration of the V_(H) region cassette(consisting of a V_(H) cassette and human C region DNA) into a pDEMexpression vector; FIG. 6B is a schematic representation of the completeH chain pDEM-VC expression vector including the V_(H) region cassetteand human C region gene; FIG. 6C is a schematic representation of acomplete L chain pDEKp-VC expression vector including the V_(L) regioncassette and human C region gene, and demonstrates the location ofinsertion of the V_(L) region cassette-human constant region fused DNAsequence into the pDEM-VC vector.

DESCRIPTION OF THE INVENTION

The method of the invention involves the preparation of apolypeptide-encoding cassette including a DNA sequence which enables thesplicing of that polypeptide-encoding segment to a secondpolypeptide-encoding DNA segment having a compatible splice sequence,thereby allowing subsequent transcription and translation of a fusionprotein. FIGS. 1 panels A and B schematically describe the methods ofthe present invention.

In FIG. 1A, a cassette is prepared including the reconstruction of the3' end of a splice donor (s.d.) site, and its attachment to the 3' endof a DNA sequence or exon encoding a first polypeptide (e.g., toxin,enzyme, lymphokine, interleukin, hormone, growth factor, or Ig domaingene such as a variable (V) region). The cassette is transfected withexpressable DNA (structural gene) for a second polypeptide (e.g., growthfactor, toxin, enzyme, lymphokine, interleukin, or Ig domain such as aconstant (C) region gene) having a compatible splice acceptor (s.a.)site at its 5' end. During the sequence of events leading up toexpression in the transfected cell, the two exons are spliced to producea mature mRNA having a 5' end encoding the first polypeptide, and a 3'end encoding another protein domain, e.g., all or part of a human Cregion. The resulting single chain polypeptide is a fusion of the firstand second polypeptides.

In FIG. 1B, a cassette is prepared including the reconstruction of the5' end of a splice acceptor site (s.a.), and its attachment to the 5'end of a DNA sequence or exon encoding a first polypeptide (e.g., toxin,enzyme, lymphokine, interleukin, growth factor, or Ig domain such as aconstant (C) region). The cassette is cotransfected with expressable DNA(structural gene e.g., a growth factor, toxin, enzyme, lymphokine,interleukin, or Ig domain such as a constant region gene) having acompatible splice acceptor site at its 5' end. During the sequence ofevents leading up to expression in the transfected cell, the two exonsare spliced to produce a mature mRNA having a 3' end encoding the firstpolypeptide, and a 5' end encoding another protein domain. The resultingsingle chain polypeptide is a fusion of the first and secondpolypeptides.

For example, an Ig V region cassette may be spliced to a C regionimmunoglobulin sequence, resulting in the expression of an intact Igmolecule, an H or L chain, or fragment thereof. Alternatively, atoxin-encoding cassette may be spliced to an Ig V region, resulting in a"magic bullet"-type therapeutic agent.

During the sequence of events leading up to expression in thetransfected cell of the fusion protein, mRNA derived from the two DNAsequences are spliced to produce a mature mRNA having a 5' end encodinga complete V_(H) or V_(L) and a 3' end encoding another protein domain,e.g., all or part of a human C region. The resulting single chainpolypeptide is a fusion of the V region and the polypeptide encoded bythe exon of the structural gene. For example, a V region cassette may bespliced to a C region. sequence, resulting in the expression of an Igmolecule, an H or L chain, or fragment thereof.

A representative case includes the construction of a chimetic bindingprotein such as an antibody. According to the method described in FIG.1A, an Ig V region cassette is prepared including a reconstruction ofthe 3' end of the V region and attachment of a splice donor site to the3' end of a V_(H) - and/or V_(L) -encoding cDNA sequence (firstpolypeptide). The modified V region cDNA is cotransfected with astructural gene for an Ig constant region (second polypeptide) having asplice acceptor site at its 5' end.

The cDNA encodes a H or L chain V region (most likely non-human, e.g.,murine) of a defined specificity, while the C region exon(s) encode theH or L chain C region of another (most likely human) Ig species.Expression of H and L constructs in a single competent host cell resultsin production of intact chimeric immunoglobulins having a desiredspecificity and, for example, an intact human constant region. Useful Vregions include the V_(H) and V_(L) domains of murine monoclonalantibody 14.18 (Mujoo et al. (1987) Cancer Res. 47: 1098-1104) whichrecognizes the disialoganglioside G_(D2) on the surface of manyneuroblastoma, melanoma, glioma, and small lung carcinoma lines andtissues. This V region can be combined with various C regions such asthat of the Ig human gamma (H) and kappa (L) chains. Alternatively, theV region may be of human origin.

To produce the V region-containing fused polypeptide, a V regioncassette is constructed and placed in an appropriate vector, togetherwith, for example, a C region-encoding DNA sequence.

The V region cassette is constructed as follows. cDNA is synthesizedfrom mRNA isolated from cells rich in V region-encoding mRNA. Usefulcell sources include lymphoid cells such as lymphocytes, myelomas andhybridomas. A number of established protocols are available for mRNAisolation and cDNA synthesis (see, e.g., Maniatis et al., ibid.). Thelongest cDNA molecules are identified, for example, with specific C_(H)(Cγ₃), C_(L) (Cκ) sequences, or other nick-translated or oligonucleotideprobes in order to detect either or both the L chain or H chain Cregion. They are then sequenced to identify the DNA sequence of the 3'end of the V region and to locate unique restriction sites within the Vregion as near as possible to the VC junction.

The next step is to synthesize an oligonucleotide which is used torecreate the sequence between the unique restriction site and splicedonor site. In the intact cDNA, the DNA sequence just 5' of the VCjunction constitutes the 5' half of the native splice donor site fromthe genome of the source cell, while the sequence just 3' of thejunction (in the C region) represents the 3' half of the native spliceacceptor site. The missing portion of each native splice site cannot bededuced from the cDNA since they were removed as part of the intronsduring mRNA synthesis in the source cell nucleus during the splicingevents.

In accordance with the invention, a DNA fragment is synthesized whichrestores the 3' side of the donor splice site, or alternatively, the 5'side of the acceptor splice site. The fragment should include enough DNA(at least about 80 nucleotide bases) to insure that efficient splicingwill occur with an appropriate splice partner sequence. In addition, itis essential that a splice donor/acceptor pair be used that results in asplice maintaining the original reading frame.

To this end, a l/a DNA sequence has been synthesized which includes anintron having parts of its sequence in common with many intronsincluding a splice donor sequence at its 3' end. This universal intron(U) (see FIGS. 1A and B) includes the splice donor sequence GTAATGTG;however, other sequences are equally as useful as splice donors andcould be utilized as well. A partial list of exemplary useful splicedonor sequences (5' and 3' portions) is shown in TABLE 1. Thesesequences are from known murine V_(H) and V_(L) genomic DNAs. The 5'ends are found at the VC junction of various murine cDNAs, while the 3'ends are removed during the splicing processes. The sequence of theuniversal intron used herein is also listed for comparison. While thesequences immediately preceding the VC junction should not differ fromthose shown (since mutations here might lead to RNA splicing problems),it should be mentioned that a 3' portion of the V region in manyexpressed Ig genes (namely, the J or joining region) undergo somaticmutation. These changes should also be included in the synthetic l/asequence, since this region may contribute to antigen binding.

                  TABLE 1                                                         ______________________________________                                        Murine Ig V(J) Region Splice Donor Sequences                                  5' end                 3' end                                                 ______________________________________                                        J.sub.K 1                                                                             GCTGGAAATCAAAC     GTAAGTAG                                           J.sub.K 2                                                                             GCTGGAAATAAAAC     GTAAGTAG                                           J.sub.K 3                                                                             GTTGGAAATAAAAC     GTAAGTAG                                           J.sub.K 4                                                                             GCTGGAGCTGAAAC     GTAAGTAC                                           J.sub.H 1                                                                             CACCGTTTCCTCAG     GTAAGCTG                                           J.sub.H 2                                                                             CACAGTCTCCTCAG     GTGAGTCC                                           J.sub.H 3                                                                             CACTGTCTCTGCAG     GTGAGTCC                                           J.sub.H 4                                                                             CACCGTCTCCTCAG     GTAAGAAT                                           (Universal Intron)     GTAATGTG                                               ______________________________________                                    

The same universal intron sequences have been used successfully withother V regions. In all cases the Ig L and H chains were efficientlyexpressed, demonstrating the generality of this process.

Recombinant DNAs comprising a truncated cDNA with restored 3' terminalfragment and donor splice site encoding a first polypeptide and areexpressed with another DNA having a splice acceptor site 5' of a regionencoding a second polypeptide. For example, a splice acceptor may bederived from genomic Ig DNA encoding all or a portion of animmunoglobulin C region. If a l/a is used which includes a universalintron having a splice acceptor site, any such known splice acceptorsite may be incorporated into the sequence. Typically, the 5' end of thesplice acceptor site includes a pyrimidine-rich region (i.e., T, C),followed by the sequence AG and the splice junction. Therefore, theuniversal intron may include such a splice acceptor sequence at its 5'end if the l/a to be used is to be linked to the 5' end of the first DNAsequence instead of the 3' end. Exemplary sequences comprising suitablesplice acceptor sites are listed in TABLE 2. These sequences are theacceptor sequences of the: (1) CH₁, (2) hinge, (3) CH₂, and (4) CH₃exons of the Cγ₁ gene. However, many other suitable splice acceptorsequences can be found in the literature.

                  TABLE 2                                                         ______________________________________                                                  5' end        3' end                                                ______________________________________                                        (1)         CTCTTGCAG       CCTCC                                             (2)         TCTCTGCAG       AGCCC                                             (3)         CTTCCTCAG       CACCT                                             (4)         GTCCTACAG       GGCAG                                             ______________________________________                                    

Upon co-expression of the two DNAs, the host cell nuclear enzymesproduce mRNA by implementing the normal splicing events, resulting in anmRNA encoding a fused protein. In the case of a chimeric bindingprotein, the mRNA may encode (5' to 3') a V_(H) or V_(L) domain, whichmay be identical to the native sequence up to the VC junction, attacheddirectly to another polypeptide such as at least a portion of at leastone C region domain, which for example, may comprise human sequences.The 3' half of the donor splice site (and any nucleotides downstream)and the 5' half of the acceptor splice site (and any nucleotidesupstream) are removed as an intron, resulting in an mRNA encoding theproperly fused protein.

Other suitable sources for C-region encloding DNA having an attached 5'splice acceptor site include human or other mammalian genomic libraries.Alternatively, a splice acceptor site having a suitable sequence may beligated to the 5' end of any desired expressible DNA sequence which maythen be fused to a DNA sequence encoding a V domain having a specificitylimited only by the specificity of available monoclonal antibodies orthose producible by known techniques.

Normally, in the case of chimeric Ig molecules, both exons are placed onthe same vector under control of a single regulating sequence. Also, itis preferred to coexpress both L and H chain constructs so that the hostcell secretes an intact fusion protein. The method requires use of ahost cell having the enzymes which recognize the DNA splice signals andeffect proper splicing.

Particular vector construction, host cell selection, transformation, andmethods of expression do not, per se, constitute an aspect of theinvention, but can be selected and implemented by skilled workers basedon personal preference and convenience. Techniques adaptable for use inthe invention are disclosed, for example, in Current Protocols inMolecular Biology (Greene Publishing Associates, 430 Fourth Street,Brooklyn, N.Y., 1989). Useful vectors include any number of knownplasmids which contain the correct signals for transcription andtranslation of the genes of interest. Enhancer elements may be present,and additional signals for polyadenylation and splicing must be presentin cases where they are not provided by the gene itself. For example,all of the signals for the expression of functionally rearranged Iggenes are present on a continuous stretch of DNA and include thetranscription promoter, the polyadenylation and termination sites, andthe splicing signals for excision of the intron sequences. Additionalinformation that must be provided by the vector is a selectable markergene. This gene must also contain the signals for expression of theselectable phenotype (usually resistance to the lethal effect of a toxicdrug such as methotrexate, for example). Therefore, if the vectorencodes both the first and second polypeptides, it is necessary that itprovide the sequence information for three separate transcription unitsin a limited amount of space.

The recombinant cassette-containing vector is transfected into anappropriate host cell. The choice of host cell line, in addition to thecriterion noted above, is based on its ability to grow in a growthmedia, preferably one that is commercially available and serum-freemedia as well as its ease of selectivity after transformation. For theproduction of chimeric antibodies, useful host cells include myelomas orhybridomas such as, for example, the murine non-Ig-producing Sp2/0 Ag 14hybridoma cell line. Useful cells are widely available in repositoriesand from commercial sources and may be isolated readily from naturalsources by those skilled in the art.

One method for introducing recombinant DNA into cells is electroporation(see, e.g., Potter et al. (1984) Proc. Natl. Acad. Sci. USA81:7161-7165), which requires specialized equipment and the availabilityof highly purified DNA. However, many different lines can be transformedusing this method if conditions are optimized for the specific celltype.

Another transfection method is protoplast (spheroplast) fusion (see,e.g., Sandri-Goldin et al. (1981) Molec. Cell. Biol. 1:743-752).Bacteria harboring the recombinant plasmid of interest are fused to thelymphold cells with a chemical agent, generally 45-50% polyethyleneglycol in a buffered, isotonic solution. This method is simple and doesnot require extensive purification of plasmid DNA. In addition, veryhigh transformation frequencies can be obtained, and the time forobtaining highly productive transfected cell clones is reduced becausethis transfection method is likely to give transfectants containingmultiple copies.

Cells which are successfully transformed with the cassette-containingvector must then be isolated from those which are not. Many methods areavailable for the selection of transfected cells. For example, theguanine phosphoribosyl transferase (gpt) and neomycin resistance markersmay be used for selection purposes in lymphoid cells. The gene encodingthe marker would be included on the V-region encoding vector. Theresistant form of dihydrofolate reductase (DHFR) can also be used forthe selection of hybridoma cell transformants as well as for subsequentamplification of the marker and flanking product genes.

The transfected cell is then cultured to express the polypeptide encodedby the cassette. Culturing may be in vitro, or in the case ofrecombinant antibodies, may be accomplished by employing otherstrategies such as in vivo culturing in ascites fluid.

Improvements in the productivity of transfected cells is possiblethrough the use of multiple subcloning steps in the presence of MTX whenthe sequences of interest are co-expressed with the DHFR marker. Forexample, after two cycles of subcloning by limiting dilution, cells areobtained that are capable of producing antibody, in spent suspensionculture medium, at levels of 35-100 μg/mL. These levels of expressionare greatly reduced if the cells are passaged in the absence of MTX. Itis possible, however, to maintain stock cultures in the presence of thedrug and then omit it from the last scale-up step. The final yields ofantibody in this case are not diminished by the omission of MTX.

In addition to open suspension culture methodologies, other scale-upperfusion technologies such as hollow fiber reactors (see, e.g., YonWedel (1987) in Commercial Production of Monoclonal Antibodies: A Guidefor Scale Up (Seaver, ed.) Marcel Dekker, Inc., New York) andmicroencapsulation (see, e.g., Rupp (1986) "Use of CellularMicroencapsulation in Large-Scale Production of Monoclonal Antibodies"in Large Scale Mammalian Cell Culture (Tolbert and Feder, eds.) AcademicPress, New York) are especially useful with the present expressionsystem. For example, it is possible to express high levels of arecombinant human antibody within microcapsules using murine hybridomatransfectants. The entrapped cell cultures can be maintained in mediumcontaining greatly reduced levels of fetal bovine serum, and in somecases, can be maintained in completely serum-free media.

Perfusion methods not only allow for higher cell densities (which maypromote cell-cell interactions and thereby reduce the serumrequirement), but may also be useful for the removal of MTX from theculture prior to purification of the antibody. This is possible usingmicroencapsulation culture wherein the semipermeable capsule membrane ismade to retain the high molecular weight antibody molecule but allow forthe diffusion out of the capsule of smaller molecules. At the end of aperfused culture run, long after cell division has stopped butproduction of antibody continues, the culture could be maintainedwithout MTX. Further removal of the drug is possible by washing anddialyzing the capsules in physiological saline prior to theirdisruption.

The invention will be further understood from the following non-limitingexamples.

EXAMPLE

Two separate H and L chain cDNA libraries are prepared according to themethod of Gubler et al. (Gene (1983) 25:263-269), herein incorporated asreference. Double-stranded, blunt-ended cDNA is synthesized frompolyadenylated polyA mRNA isolated from the murine hybridoma cell line,14.18.

A double-stranded polylinker with the sequence: ##STR1## is then ligatedto the cDNA. This linker serves several purposes. The first allows theblunt-ended cDNA to be cloned into the EcoRI site of λgt10 phage DNA viathe AATT sticky-end sequence. Note that only the 5' blunt end has beenphosphorylated for ligation to the blunted cDNA. By not phosphorylatingthe 5' EcoRI sticky end, polymerization of the linkered cDNA does notoccur and subsequent enzymatic digestion after linker ligation isunnecessary. This, in turn, makes it unnecessary to methylate internalEcoRI sites which would otherwise be cut in the process. The 5'phosphorylated sticky end provided by the EcoRI digested λgt10 DNA issufficient for ligation and subsequent cloning of the cDNA in the phagehost.

Another function of this linker sequence is to provide XhoI (CTCGAG) andSalI (GTCGAC) sites for subsequent manipulations. These sites rarelyoccur in murine Ig cDNAs and thus provide unique restriction sites atthe 5' and 3' ends. Since both sites provide the same 5' overhangsequence upon restriction (TCGA), either can be used in expressionvectors with XhoI cloning sites. If one site happens to appear in thecDNA sequence, the chances are small that the second site would also bepresent.

The linkered cDNA is then fractionated by polyacrylamide gelelectrophoresis (PAGE) to enrich for full-length L and H chain cDNAs.Two fractions of cDNA corresponding to full-length L chain (900-1100 bp)and H chain (1400-1600 bp) can be separately isolated. The DNA in eachgel fraction is separately eluted and ligated to EcoRI-digested λgt10DNA. Following in vitro packaging with commercially available packagingmix (Stratagene, San Diego, Calif.), recombinant phage is plated andscreened by filter hybridization using various C region probes.

Ten phage clones from each screening are analyzed further by restrictionanalysis using ECoRI. This is most rapidly achieved by preparingsmall-scale phage lysates which provide enough DNA for restrictiondigest and determination of the length of the cDNA insert. Clones thatappear to be full-length are labelled with radioactivity, e.g. at theunique EcoRI sites at the 5' and 3' ends (relative to the original mRNApolarity). Following digestion with a second enzyme, the clones aresequenced by the method of Maxam and Gilbert (Meth. Enzymol. (1980)65:499-559), herein incorporated by reference, and screened with probesfor murine Cγ₃ and Cκ sequences.

The longest H cDNA sequence is shown in FIG. 2. The H chain cloneappears to be very close to full-length if it is assumed that the secondATG in the sequence represents the true initiation codon. Although bothATG codons are in the correct reading frame, the first is probably tooclose to the 5' end of the mRNA for efficient initiation and, iftranslated, would encode an uncharacteristically long leader sequence.The use of the second ATG codon would result in the synthesis of a verytypical Ig leader sequence of 19 amino acids. Because additional 5'untranslated sequences are to be added to this cDNA in the expressionvector (see below), the first ATG would no longer be at the 5' end ofthe resulting fusion mRNA, thereby increasing the likelihood of thetranslation of the aberrant leader sequence. To avoid this problem, thecDNA sequence is truncated by limited Ba131 exonuclease treatment. Tothe resulting modified cDNA clone (H2c) is then attached an XhoI linker(see FIG. 2). The expression of this cDNA should result in an mRNAencoding a normal Ig leader sequence. The remainder of the V_(H) portionof the cloned cDNA appears to be a normal, functional variable region.

Examination of the L chain clone (FIG. 3) shows a typical L chainsequence encoding a 19 amino acid leader followed by sequences that arehighly homologous to the anti-GAT family of V.sub.κ genes. The murine κC region begins with the arginine residue at position 114.

Reconstruction of the donor splice sites on the 3' ends of the clonedcDNAs for V_(H) and for V_(L) is carried out as shown in FIGS. 4A to Eand 5A to E. The first step is to sequence both V_(H) and V_(L) regions,and then to identify unique restriction sites near the junctions of theV and C regions (see FIGS. 2 and 3).

The sequence between the unique restriction site and the original splicedonor site in the genomic V gene segment is then recreated using theuniversal intron sequence previously shown in TABLE 1. For convenience,the oligonucleotides used to reconstruct the intron sequence are clonedas a SnaeBl to HindIII fragment (the latter site was not originally partof the intron). The SnaeBl site can then be cut to produce a blunt endbeginning with GTA (the bases that reconstitute the splice signal) whilethe HindIII site serves as the 3' cloning site for insertion of thecassette into the expression vector.

The small linker portions of the V regions shown in panels A-E of FIGS.4A to E and 5A to E are synthesized by the hybridization and ligation ofoverlapping oligonucleotides to produce fragments with the appropriate"sticky" 5' end, as well as a blunt 3' end. Each synthetic l/a sequenceis then cloned into a plasmid vector for sequence verification. In somecases the 5' cloning site may not be unique or present in a convenientplasmid. In these cases, additional restriction sites can be added tothe 5' end of the synthetic DNA for cloning purposes. After DNAsequencing, the l/a fragments are ligated to the remaining portions ofthe V regions. The complete V_(H) and V_(K) region cassettes are thencloned as XhoI to HindIII fragments.

The mammalian cell expression vector used for the expression of thechimeric 14.18 antibody is shown in FIG. 6 panels A-C. It was derivedfrom the pDEM vector (FIG. 6A), which has been used to express cDNAsequences in lymphold cells. This vector was designed as follows. At theleft end of the schematic linear representation there is the selectablemarker gene, DHFR, which is oriented so that transcription would proceedfrom right to left. This transcription unit is composed of SV40regulatory signals (enhancer and promoter), a cDNA encoding theresistant form of mouse DHFR, and an SV40 polyA site.

Adjacent to this gene (to the right of the SalI site) is thetranscription unit for the cDNA of interest. The first block of sequencerepresents the Ig H chain enhancer (E.sub.μ), the sequence that isnormally involved in the active transcription of Ig H-chain genes. Theenhancer sequence is followed by the promoter for the murinemetallothionein (MT) gene. Many other promoters can be used in thisposition including those from Ig genes, as in the L chain transcriptionunit shown in FIG. 6C. A synthetic restriction site (for XhoI) is placedin the 5' untranslated region of the MT sequence so that CDNAs can beinserted at this position and be transcribed as fusion mRNAs from the MTpromoter. In this case it is necessary that the CDNA contain its owntranslation initiation codon since it is not provided by the vector.Beyond the XhoI site (after the insertion site for cDNAs) is a segmentderived from the 3' untranslated region of the murine Cκ region thatcontains the polyA addition site. A cDNA expressed from thistranscription unit would then be composed of the 5' untranslatedsequence from the MT mRNA, the specific coding sequence of interest, andthe 3' untranslated sequence normally found on murine κ mRNAs.

In the present example of chimeric antibodies, some of the components ofthis plasmid are superfluous, namely the 3' untranslated sequence andthe polyA site from the κ gene segment. Instead of these components, theV region cassette and the human C region gene segment between the XhoIand SalI sites of plasmid pDEM (see FIG. 6A) are inserted. Since the Cregion gene already provides the 3' untranslated sequences and the polyAsite, the only signals to be used from the plasmid are those necessaryfor the transcription of the cDNA (enhancer and promoter). The resultingplasmid is shown in FIG. 6B.

A similar construct is made for the L chain, but in this case theplasmid has a guanine phosphoribosyltransferase (gpt) selectable markergene not present in the final vector, and the C region is that of thehuman κ gene. The promoter used for L chain cDNA expression is derivedfrom a Vκ gene, and a unique XhoI site is placed in the 5' untranslatedregion for fusion to the cDNA. This latter construct is designed so thatthe entire L chain transcription unit is flanked by SalI sites, while inthe case of the H chain vector, only a single SalI site is retained. Inthis way it is possible to excise the entire L chain transcription unit(about 3 kb), and then to insert it into the unique SalI site of theH-chain expression vector (FIG. 5C).

Another important design feature of the Ig expression vector involvesthe use of the E.sub.μ enhancer for the expression of both H and Lchains. Since this enhancer is much more powerful than the L chaincounterpart (at least in transfection experiments), a higher and morebalanced level of expression of both chains is possible. The L chaintranscription unit also contains some additional sequences between theSalI site and the E.sub.μ sequence. This sequence is derived from the λ₁L chain promoter, and serves to attenuate the bi-directional enhancingeffect of E.sub.μ in one direction (away from the L chain transcriptionunit) without affecting enhancement of transcription of the cDNA (EPO87/300658.9). When the L chain transcription unit is inserted into theexpression vector as shown in FIG. 6 panels A-C, this sequence preventsthe overexpression of the adjacent marker gene by blocking the enhancereffect in that direction. In this way it is hoped that transfectantswill produce more of the transfected protein of interest relative to theproduct of the marker gene.

The murine non-Ig-producing hybridoma cell line, Sp2/0 Ag 14 cells aretransfected with the chimeric Ig plasmid construct described above usinga modified protoplast fusion method (Gillies et al. (1983) Cell33:717-728), herein incorporated by reference.

After plating in 96-well culture dishes, selection medium (Dulbecco'smodified Eagle's medium (DMEM) with 10% fetal bovine serum) containingMTX (0.1 μM) is added. Cells are fed with 50% culture medium replacementat 3-4 day intervals for a total of three feedings. Colonies ofMTX-resistant cells appear in 10-14 days.

At this point culture supernatants are assayed for secreted humanantibody determinants by an ELISA assay. Nearly all the MTX-resistantcolonies secrete significant levels of human antibody into the medium.

After identifying the clones which secrete the most antibody, theexpression level is enhanced by increasing the concentration of MTX inthe medium. The cells quickly adapt to dramatic increases inconcentration (as much as 10-fold in a single step) with little or nocell death. The level of MTX is increased from 0.1 to 10 μM in a periodof about three weeks. During this time, the concentration of antibodyincreases from a range of 2 to 8 μg/mL (at 0.1 μM MTX) to a range of 10to 40 μg/mL of spent culture medium (at 10 μM MTX). Levels in excess of100 μg/mL of spent culture medium have been obtained using this andother similar constructs embodying the invention.

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The presentembodiments are therefore to be considered in all respects asillustrative and not restrictive, the scope of the invention beingindicated by the appended claims rather than by the foregoingdescription, and all changes which come within the meaning and range ofequivalency of the claims are therefore intended to be embraced therein.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 23                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A ) NAME/KEY: miscfeature                                                    (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: /note="GAP: 14...15                                    sequence: Jk1"                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GCTGGAAATCAAACGTAAGTAG22                                                      (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: miscfeature                                                     (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: /note="GAP: 14...15                                    sequence: Jk2"                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GCTGGAAATAAAACGTAAGTAG 22                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: miscfeature                                                     (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: /note="GAP: 14...15                                     sequence: Jk3"                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GTTGGAAATAAAACGTAAGTAG22                                                      (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                  (A) NAME/KEY: miscfeature                                                    (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: /note="GAP: 14...15                                    Sequence: Jk4"                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GCTGGAGCTGAAACGTAAGTAC22                                                      (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                      (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: miscfeature                                                     (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: /note="GAP: 14...15                                    Sequence: Jh1"                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CACCGTTTCCTCAGGTAAGCTG 22                                                     (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: miscfeature                                                     (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: /note="GAP: 14...15                                     Sequence: Jh2"                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CACAGTCTCCTCAGGTGAGTCC22                                                      (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: miscfeature                                                     (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: /note="GAP: 14...15                                    Sequence: Jh3"                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CACTGTCTCTGCAGGTGAGTCC22                                                      (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: miscfeature                                                     (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: /note="GAP: 14...15                                    Sequence: Jh4"                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       CACCGTCTCCTCAGGTAAGAA T22                                                     (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 base pairs                                                      (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: intron                                                          (B) LOCATION: 1..8                                                             (D) OTHER INFORMATION: /note="Sequence: Universal                            Intron"                                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GTAATGTG8                                                                     (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: miscfeature                                                     (B) LOCATION: 1..14                                                           (D) OTHER INFORMATION: /note="GAP: 9...10                                     Sequence:                                                                     Splice Acceptor 1"                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      CTCTTGCAGCCTCC14                                                              (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 14 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: miscfeature                                                     (B) LOCATION: 1..14                                                           (D) OTHER INFORMATION: /note="GAP: 9...10                                     Sequence:                                                                     Splice Acceptor 2"                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      TCTCTGCAGAGCCC14                                                              (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: miscfeature                                                      (B) LOCATION: 1..14                                                          (D) OTHER INFORMATION: /note="GAP: 9...10 Splice                              Acceptor 3"                                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      CTTCCTCAGCACCT14                                                              (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: miscfeature                                                     (B) LOCATION: 1..14                                                           (D) OTHER INFORMATION: /note="GAP: 9...10                                     Sequence:                                                                     Splice Acceptor 4"                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GTCCTACAGGGCAG 14                                                             (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: miscfeature                                                     (B) LOCATION: 1..16                                                           (D) OTHER INFORMATION: /note="Sequence: Polylinker 5'"                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      AATTCCTCGAGTCGAC16                                                            (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                  (A) NAME/KEY: miscfeature                                                    (B) LOCATION: 1..12                                                           (D) OTHER INFORMATION: /note="Sequence: Polylinker 5'"                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GTCGACTCGAGG12                                                                (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 499 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: miscfeature                                                     (B) LOCATION: 1..499                                                          (D) OTHER INFORMATION: /note="Leader Sequence: 41...97                        Sequence: 14.18 H Chain"                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      AATGTCCTCTCCACAGTCCCTGAAGACACTGACTATAACTATGGGATGG ACCTGGATCTT60               TATTTTAATCCTGTCGGTAACTACAGGTGTCCACTCTGAGGTCCAACTGCTGCAGTCTGG120               ACCTGAGCTGGAGAAGCCTAGCGCTTCAGTGATGATATCCTGCAAGGCTTCTGGTTCCTC180               ATTCACTGGCTACAACATGAACTGGGTGAGGCA GAACATTGGAAAGAGCCTTGAATGGAT240              TGGAGCTATTGATCCTTACTATGGTGGAACTAGCTACAACCAGAAGTTCAAGGGCAGGGC300               CACATTGACTGTAGACAAATCGTCCAGCACAGCCTACATGCACCTCAAGAGCCTGACATC360               TGAGGACTCTGCAGTC TATTACTGTGTAAGCGGAATGGAGTACTGGGGTCAAGGAACCTC420              AGTCACCGTCTCCTCAGCTACAACAACAGCCCCATCTGTCTATCCCTTGGTCCCTGGCTG480               CAGTGACACATCCGGATCC499                                                        (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 489 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: miscfeature                                                     (B) LOCATION: 1..489                                                          (D) OTHER INFORMATION: /note="Leader Sequence: 40...96                        Sequence: 14.18 L Chain"                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      CAGTCTCCTCAGGCTGTCTCCTCAGGTTGCCTCCTCAAAATGAAGTTGCCTGTTAGGCTG60                TTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTGATGTTGTGATGACCCAGACTCCA120               CTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGC AGATCTAGTCAGAGT180              CTTGTACACCGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCT240               CCAAAGCTCCTGATTCACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGT300               GGCAGTGGATCAGGGACAGATTTCACACTC AAGATCAGCAGAGTGGAGGCTGAGGATCTG360              GGAGTTTATTTCTGTTCTCAAAGTACACATGTTCCTCCGCTCACGTTCGGTGCTGGGACC420               AAGCTGGAGCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGT480               GAGCAGTTA 489                                                                 (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..57                                                            (D) OTHER INFORMATION: /note="Splice Site: 43...44                           Sequence: H Chain VC Junction"                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      ATGGAGTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCTACA48                            MetGluTyrTrpGlyGlnGlyThrSerValThrValSerSerAlaThr                              1 51015                                                                       ACAACAGCC57                                                                   ThrThrAla                                                                     (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      MetGluTyrTrpGlyGlnGlyThrSerValThrValSerSerAlaThr                              151015                                                                        ThrThrAla                                                                     (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 70 base pairs                                                     (B) TYPE: nucleic acid                                                        ( C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: miscfeature                                                     (B) LOCATION: 52..53                                                          (D) OTHER INFORMATION: /note="Splice Site: 40...41                            GAP: 52...53 Sequence: H Chain l/a"                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      GAGTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGGTAAGTGTG TCAGGTAATGT60               CACTAAGCTT70                                                                  (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 66 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                            (B) LOCATION: 1..66                                                           (D) OTHER INFORMATION: /note="Splice Site: 52...53                            Sequence: L Chain VC Junction"                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      ACACATGTTCCTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTG48                            ThrHisValProProLeuThrPheGlyA laGlyThrLysLeuGluLeu                             151015                                                                        AAACGGGCTGATGCTGCA66                                                          LysArgAlaAspAlaAla                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 ( A) LENGTH: 22 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      ThrHisValProProLeuThrPheGlyAlaGlyThrLysLeuGluLeu                              151015                                                                        LysArgAlaAspAla Ala                                                           20                                                                            (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 77 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: miscfeature                                                     (B) LOCATION: 1..77                                                           (D) OTHER INFORMATION: /note="Splice Site: 50...51                             GAP: 59...60 Sequence: L Chain l/a"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      ACACGTTCCTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGTAAGTGTGG60                GTAATGTCACTAAGCTT77                                                       

We claim:
 1. A method of producing a chimeric antibody having apredetermined specificity, said method comprising the steps of:(a)providing a variable (V) region cassette comprisinga first nucleic acidhaving a sequence that encodes a V region having a predeterminedspecificity, said first nucleic acid being covalently linked through its3' end to a second nucleic acid having a sequence that encodes a splicedonor site,wherein a portion of the 3' end of said first nucleic acid,together with said second nucleic acid, comprises a syntheticoligonucleotide; (b) ligating said V region cassette with a thirdnucleic acid having a sequence therein that encodes a humanimmunoglobulin constant (C) region, said third nucleic acid furthercomprising a 5' splice acceptor site at its 5' end, compatible with saidsynthetic splice donor site on said V region cassette; (c) transfectingsaid ligated nucleic acid into a eucaryotic host cell, and (d) culturingsaid transfected host cell to produce said chimeric antibody having saidpredetermined specificity and antigen binding activity.
 2. The method ofclaim 1 wherein said first nucleic acid provided in step (a) encodes animmunoglobulin light chain V region.
 3. The method of claim 2 whereinsaid first nucleic acid encodes a non-human immunoglobulin V region. 4.The method of claim 3 wherein said first nucleic acid encoding animmunoglobulin V region encodes a murine V region.
 5. The method ofclaim 1 wherein said first nucleic acid provided in step (a) encodes animmunoglobulin heavy chain V region.